Enzymatic properties of single-chain and two-chain forms of a Lys158----Glu158 mutant of urokinase-type plasminogen activator.

Abstract

Recombinant single-chain urokinase-type plasminogen activator (rscu-PA), in which the plasmin-sensitive peptide bond Lys158-Ile159 is destroyed by site-specific mutagenesis of Lys158 to Glu (rscu-PA-Glu158), is quantitatively converted to two-chain urokinase-type plasminogen activator (rtcu-PA-Glu158) by treatment with endoproteinase Glu-C (Staphylococcus aureus V8 proteinase). The catalytic efficiency (k2/Km) of rscu-PA-Glu158 for the activation of plasminogen is 20 times lower (0.0001 microM-1 s-1) than that of rscu-PA (0.002 microM-1 s-1). In contrast, rtcu-PA-Glu158 has very similar properties to rtcu-PA obtained by digestion of rscu-PA with plasmin, including binding to benzamidine-Sepharose, catalytic efficiency for the activation of plasminogen (0.035 microM-1 s-1 versus 0.046 microM-1 s-1) and fibrinolytic activity in an in vitro plasma clot lysis system. It is concluded that the amino acid in position 158 is a main determinant of the functional properties of single-chain urokinase-type plasminogen activator but not of the two-chain form.